This is a RO1 application by a new investigator. The overall goal of this project is to identify components of impaired hypoglycemic counterregulation as well as to identify and test potential new targets for therapeutic intervention. Common findings in patients with type 1 and advanced type 2 diabetes are impaired glucose sensing, hypoglycemia unawareness and impaired counterregulation. These pathological processes exacerbate the frequency and severity of insulin-induced hypoglycemic episodes making hypoglycemia the rate limiting step in the glycemic management of diabetes. Preliminary results in this application demonstrate that insulin, in addition to acutely lowering blood glucose levels, has a major direct effect in the brain to independently modulate both glucose sensing and the sympathoadrenal response to hypoglycemia. The overall hypothesis in this proposal is that modulation of insulin-sensitive therapeutic targets in the hypothalamus of the central nervous system can improve the cognitive and counterregulatory responses to hypoglycemia in experimental animals. The use of a regional hypothalamic RT-PCR technique to identify key genes and combined patterns of hypothalamic gene expression will help to identify molecular pathology in animal models of impaired hypothalamic glucose sensing and impaired counterregulation. The specific aims to be addressed in this research proposal are to: (1) dissect the potential role of insulin action in the central nervous system (CNS) in regulating the counterregulatory response to hypoglycemia; (2) determine the mechanisms by which insulin may regulate hypothalamic glucose sensing via regulation of glucose transporters and/or glucokinase and correlate these insulin-modulated changes in glucose sensing neurons with neuronal functional activity under conditions of hypoglycemia; (3) explore the role of insulin in altering cognitive function during acute hypoglycemia in models of increased or decreased CNS insulin action and differentiate the cognitive from the counterregulatory responses; and (4) examine the pattern of regional hypothalamic gene expression uniquely attributable to insulin-induced recurrent hypoglycemia with the use of RT-PCR and correlate alterations in gene expression with impaired glucose sensing and impaired hypoglycemic counterregulation.